The effective application of gene therapy to human hematopoietic diseases continues to be hampered by the inability to introduce genetic material into a sufficiently large proportion of hematopoietic stem cells. Efficient transduction of murine hematopoietic stem cells has been achieved by treating mice with 5-fluorouracil (5-FU) or with the combination of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) to elicit stem cell proliferation before collecting cells. In large animal models and humans it is has not yet been possible to transduce a sufficiently high proportion of marrow repopulating stem cells to potentially effect a therapeutic benefit. We hypothesize that, as in mice, the induction of proliferation of hematopoietic stem cells in humans will be required for effective gene transduction by retroviral vectors. Using baboons as a model, we have demonstrated that lymphohematopoietic stem cells express the CD34 antigen, that stem cells are mobilized into the peripheral blood by treatment with SCF or with SCF plus G-CSF, and that the transduction efficiency of marrow CD34+ progenitors by retroviral vectors is increased 10-fold 6 days after 5-FU treatment. We hypothesize that perturbing hematopoiesis in baboons with cytokine combinations and/or cytotoxic drugs will elicit CD34+ stem cell populations more susceptible to transduction by retroviral and possibly AAV vectors. Therefore, the SPECIFIC AIMS of this project are to 1) determine if treatment of baboons with SCF and G-CSF alone or in combination, or 5-FU alone or in combination with SCF and G-CSF will increase the ability to transduce CD34+ stem cells from bone marrow and peripheral blood using murine amphotropic retroviral vectors, and to compare these results with those obtained with CD34+ cord blood cells. 2) Determine if CD34+ stem cells from marrow, mobilized blood, and cord blood are more susceptible to transduction by gibbon ape leukemia virus and AAV vectors as compared to murine amphotropic vectors. 3) Determine if genetically altered CD34+ cells from different sources differ in their capacity to reconstitute lymphohematopoiesis in vivo. These studies will therefore define the conditions necessary to achieve effective gene transfer into hematopoietic stem cells in baboons and will provide further insights directly applicable to gene therapy in humans.